The ultimate purpose of this research proposal is to determine the role of feedback inhibition of Insulin Receptor Substrate (IRS-1) in insulin resistance. Differentiated 3T3-L1 adipocytes, treated with the phosphatase inhibitor okadate acid (OKA), have been shown to exhibit insulin resistance, accompanied by decreased insulin stimulated tyrosine phosphorylation of IRS-1 and an increase serine/threonine kinase activity capable of phosphorylating IRS-1. We are attempting to identify the serine threonine kinase activity, first by examining the role of two likely candidate proteins, MAP kinase and Protein Kinase C, and by attempting to purify a novel kinase. We will attempt to establish the biological relevance of this kinase activity by examining the effect of expressing IRS-1 mutants, lacking one or more of the potential ser/thr phosphorylation sites, on the OKA induced insulin resistance. We hope to establish a direct correlation between ser/thr phosphorylation of IRS-1 and a decrease in its tyrosine phosphorylation by insulin stimulated IR, decreased PI-3 kinase binding and decreased glucose uptake.